Revision 1
Cell Signaling Technology

Orders: 877-616-CELL (2355) [email protected]

Support: 877-678-TECH (8324)

Web: [email protected] cellsignal.com

3 Trask LaneDanversMassachusetts01923USA
For Research Use Only. Not for Use in Diagnostic Procedures.
Product Includes Product # Quantity Mol. Wt Isotype/Source
Ubiquitin Antibody 3933 40 µl Rabbit 
UBE1a Antibody 4890 40 µl 117 kDa Rabbit 
UBC3 Antibody 4997 40 µl 32 kDa Rabbit 
UbcH5C (D60E2) Rabbit mAb 4330 40 µl 14 kDa Rabbit IgG
UBE2L3 (D5G1) Rabbit mAb 8721 40 µl 18 kDa Rabbit IgG
UBE2N/Ubc13 (D2A1) Rabbit mAb 6999 40 µl 17 kDa Rabbit IgG
Anti-rabbit IgG, HRP-linked Antibody 7074 100 µl Goat 

Please visit cellsignal.com for individual component applications, species cross-reactivity, dilutions, protocols, and additional product information.

Description

The Ubiquitin Activation (E1, E2 Enzymes) Antibody Sampler Kit provides an economical means to study ubiquitin activation and conjugation. This kit contains enough primary antibody to perform four western blots per primary.

Storage

Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA, 50% glycerol and less than 0.02% sodium azide. Store at –20°C. Do not aliquot the antibody.

Background

Ubiquitin is a conserved polypeptide unit that plays an important role in the ubiquitin-proteasome pathway. Ubiquitin can be covalently linked to many cellular proteins for degradation by the 26S proteasome. Three components are involved in the target protein-ubiquitin conjugation process. Ubiquitin is first activated by forming a thioester complex with the ubiquitin-activating enzyme (UBE1 or E1). The activated ubiquitin is subsequently transferred to the ubiqutin-carrier protein (conjugating enzyme) E2, and then from E2 to ubiqutin ligase E3 for final delivery to the epsilon-NH2 of the target protein lysine residue (1-3). The ubiquitin-proteasome pathway has been implicated in a wide range of normal biological processes and in disease-related abnormalities. Several proteins such as IκB, p53, cdc25a, and Bcl-2 have been shown to be targets for the ubiquitin-proteasome process as part of the regulation of cell cycle progression, differentiation, cell stress response, and apoptosis (4-7). UBC3, the mammalian ortholog of yeast cdc34, and UBC3B, a UBC3 family member, are E2 ubiquitin-carrier proteins. UBC3, in concert with SCF-Skp2 (Skp1, Cullin, F-box protein/Skp2) complex, mediates cell cycle progression from G1 to S phase by targeting the CDK inhibitor p27 for proteolysis (8). UBC3B, in concert with SCFb-Trcp (Skp1, Cullin and F-box protein/b-Trcp) complex, mediates degradation of β-catenin (9). UbcH5C is a universally expressed E2 ubiquitin conjugating enzyme and member of the UbcH5 family that also includes UbcH5A and UbcH5B (10). Evidence suggests that UbcH5 plays an important role in regulating a number of signaling pathways by catalyzing the ubiquitination of key target proteins, including p53, PCNA, the IκB kinase proein NEMO, and the apoptosis inhibitor BRUCE (11-14). UBE2L3, also commonly referred to as UBCH7, is a ubiquitin-conjugating enzyme that has been linked to the ubiquitination of numerous substrates via its interaction with protein-ubiquitin E3 ligases, such as NEDD4 (15), E6AP (16), Parkin (17), c-Cbl (18), and Triad1 (19,20). UBE2N/Ubc13 is a ubiquitin-E2-conjugating enzyme that catalyzes K63-linked polyubiquitin chain formation (21,22). UBE2N forms a heterodimer with MMS2 or Uev1A to exert its E2 ligase function. The UBE2N/MMS2 and UBE2N/Uev1A heterodimers catalyze different modes of target protein ubiquitination to mediate various signaling pathways (23-25) including DNA damage and recombination, p53 and check point control, cell cycle (26-30), immunoreceptor signaling (31,32), and endocytosis (33).

  1. Ciechanover, A. (1998) EMBO J 17, 7151-60.
  2. Hochstrasser, M. (2000) Nat Cell Biol 2, E153-7.
  3. Hochstrasser, M. (2000) Science 289, 563-4.
  4. Bernardi, R. et al. (2000) Oncogene 19, 2447-54.
  5. Aberle, H. et al. (1997) EMBO J 16, 3797-804.
  6. Salomoni, P. and Pandolfi, P.P. (2002) Nat Cell Biol 4, E152-3.
  7. Jesenberger, V. and Jentsch, S. (2002) Nat Rev Mol Cell Biol 3, 112-21.
  8. Pagano, M. et al. (1995) Science 269, 682-5.
  9. Semplici, F. et al. (2002) Oncogene 21, 3978-87.
  10. Jensen, J.P. et al. (1995) J Biol Chem 270, 30408-14.
  11. Saville, M.K. et al. (2004) J Biol Chem 279, 42169-81.
  12. Zhang, S. et al. (2008) Cell Cycle 7, 3399-404.
  13. Tang, E.D. et al. (2003) J Biol Chem 278, 37297-305.
  14. Qiu, X.B. et al. (2004) EMBO J 23, 800-10.
  15. Anan, T. et al. (1998) Genes Cells 3, 751-63.
  16. Huang, L. et al. (1999) Science 286, 1321-6.
  17. Shimura, H. et al. (2001) Science 293, 263-9.
  18. Yokouchi, M. et al. (1999) J Biol Chem 274, 31707-12.
  19. Marteijn, J.A. et al. (2009) Leukemia 23, 1480-9.
  20. Marteijn, J.A. et al. (2005) Blood 106, 4114-23.
  21. Herrmann, J. et al. (2007) Circ Res 100, 1276-91.
  22. Wilkinson, K.D. et al. (2005) EMBO Rep 6, 815-20.
  23. Hofmann, R.M. and Pickart, C.M. (1999) Cell 96, 645-53.
  24. Deng, L. et al. (2000) Cell 103, 351-61.
  25. Andersen, P.L. et al. (2005) J Cell Biol 170, 745-55.
  26. Zhao, G.Y. et al. (2007) Mol Cell 25, 663-75.
  27. Kolas, N.K. et al. (2007) Science 318, 1637-40.
  28. Laine, A. et al. (2006) Mol Cell Biol 26, 8901-13.
  29. Huen, M.S. et al. (2008) Mol Cell Biol 28, 6104-12.
  30. Loring, G.L. et al. (2008) Cell Cycle 7, 96-105.
  31. Yamamoto, M. et al. (2006) Nat Immunol 7, 962-70.
  32. Yamamoto, M. et al. (2006) J Immunol 177, 7520-4.
  33. Duncan, L.M. et al. (2006) EMBO J 25, 1635-45.

Background References

    Trademarks and Patents

    Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
    All other trademarks are the property of their respective owners. Visit cellsignal.com/trademarks for more information.

    Limited Uses

    Except as otherwise expressly agreed in a writing signed by a legally authorized representative of CST, the following terms apply to Products provided by CST, its affiliates or its distributors. Any Customer's terms and conditions that are in addition to, or different from, those contained herein, unless separately accepted in writing by a legally authorized representative of CST, are rejected and are of no force or effect.

    Products are labeled with For Research Use Only or a similar labeling statement and have not been approved, cleared, or licensed by the FDA or other regulatory foreign or domestic entity, for any purpose. Customer shall not use any Product for any diagnostic or therapeutic purpose, or otherwise in any manner that conflicts with its labeling statement. Products sold or licensed by CST are provided for Customer as the end-user and solely for research and development uses. Any use of Product for diagnostic, prophylactic or therapeutic purposes, or any purchase of Product for resale (alone or as a component) or other commercial purpose, requires a separate license from CST. Customer shall (a) not sell, license, loan, donate or otherwise transfer or make available any Product to any third party, whether alone or in combination with other materials, or use the Products to manufacture any commercial products, (b) not copy, modify, reverse engineer, decompile, disassemble or otherwise attempt to discover the underlying structure or technology of the Products, or use the Products for the purpose of developing any products or services that would compete with CST products or services, (c) not alter or remove from the Products any trademarks, trade names, logos, patent or copyright notices or markings, (d) use the Products solely in accordance with CST Product Terms of Sale and any applicable documentation, and (e) comply with any license, terms of service or similar agreement with respect to any third party products or services used by Customer in connection with the Products.

    Revision 1
    #8645

    Ubiquitin Activation (E1, E2 Enzymes) Antibody Sampler Kit

    Ubiquitin Activation (E1, E2 Enzymes) Antibody Sampler Kit: Image 1 Expand Image
    Western blot analysis of extracts from HeLa and NIH/3T3 cells, untreated or treated with the proteasome inhibitor MG132 (10 µM for 90 minutes), using Ubiquitin Antibody.
    Ubiquitin Activation (E1, E2 Enzymes) Antibody Sampler Kit: Image 2 Expand Image
    Western blot analysis of extracts from various cell lines using UbcH5C (D60E2) Rabbit mAb.
    Ubiquitin Activation (E1, E2 Enzymes) Antibody Sampler Kit: Image 3 Expand Image
    Western blot analysis of extracts from K562 and SK-N-MC cells using UBE1a Antibody.
    Ubiquitin Activation (E1, E2 Enzymes) Antibody Sampler Kit: Image 4 Expand Image
    Western blot analysis of extracts from Jurkat, CAD and C6 cells using UBC3 Antibody.
    Ubiquitin Activation (E1, E2 Enzymes) Antibody Sampler Kit: Image 5 Expand Image
    Western blot analysis of extracts from 293T cells, either mock transfected or transfected with a Myc/DDK-tagged cDNA expression construct encoding full-length human UBE2N, UBE2D1, UBE2D2, UBE2D3, or UBE2D4, using UBE2N/Ubc13 (D2A1) Rabbit mAb (upper) and DYKDDDDK Tag Antibody (Binds to same epitope as Sigma's Anti-FLAG® M2 Antibody) #2368 (lower).
    Ubiquitin Activation (E1, E2 Enzymes) Antibody Sampler Kit: Image 6 Expand Image
    After the primary antibody is bound to the target protein, a complex with HRP-linked secondary antibody is formed. The LumiGLO® is added and emits light during enzyme catalyzed decomposition.
    Ubiquitin Activation (E1, E2 Enzymes) Antibody Sampler Kit: Image 7 Expand Image
    Western blot analysis of extracts from various cell lines using UBE2L3 (D5G1) Rabbit mAb.
    Ubiquitin Activation (E1, E2 Enzymes) Antibody Sampler Kit: Image 8 Expand Image
    Immunohistochemical analysis of paraffin-embedded human breast carcinoma using Ubiquitin Antibody in the presence of control peptide (left) or antigen-specific peptide (right).
    Ubiquitin Activation (E1, E2 Enzymes) Antibody Sampler Kit: Image 9 Expand Image
    Immunohistochemical analysis of paraffin-embedded glioblastoma using UBE1a Antibody.
    Ubiquitin Activation (E1, E2 Enzymes) Antibody Sampler Kit: Image 10 Expand Image
    Western blot analysis of extracts from various cell lines using UBE2N/Ubc13 (D2A1) Rabbit mAb.
    Ubiquitin Activation (E1, E2 Enzymes) Antibody Sampler Kit: Image 11 Expand Image
    Immunohistochemical analysis of paraffin-embedded human lung carcinoma using Ubiquitin Antibody.
    Ubiquitin Activation (E1, E2 Enzymes) Antibody Sampler Kit: Image 12 Expand Image
    Immunohistochemical analysis of paraffin-embedded human lung carcinoma using UBE1a Antibody.
    Ubiquitin Activation (E1, E2 Enzymes) Antibody Sampler Kit: Image 13 Expand Image
    Immunohistochemical analysis of paraffin-embedded mouse brain using UBE1a Antibody in the presence of control peptide (left) or antigen specific peptide (right).
    Ubiquitin Activation (E1, E2 Enzymes) Antibody Sampler Kit: Image 14 Expand Image
    Confocal immunofluorescent analysis of SK-N-MC cells using UBE1a Antibody (green). Actin filaments have been labeled with Alexa Fluor® 555 phalloidin (red).
    Ubiquitin Activation (E1, E2 Enzymes) Antibody Sampler Kit: Image 15 Expand Image
    Flow cytometric analysis of K562 cells using UBE1a Antibody (blue) compared to a nonspecific negative control antibody (red).